Many proteins require glycosylation for their biological function. Often, the terminal, “capping,” carbohydrates of glycosylic chains are sialic acid residues. Sialic acids comprise a family of N- and O-linked neuraminic acids. N-linked sialic acids are formed by linking acetyl or glycolyl moieties to the amino residue of neuraminic acid, forming N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc), respectively. If the amino group of neuraminic is substituted with a hydroxyl moiety, this yields 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN). O-linked sialic acids are formed by the substitutions of one or more of the hydroxyl groups of Neu5Ac, Neu5Gc, or KDN with methyl, acetyl, lactoyl, sulfate, or phosphate groups. Accordingly, a large and diverse population of sialic acids exists.
Further, there is considerable interest in analyzing protein sialylation, in general, because of the numerous biological functions attributed to these modifications. Sialylation can be important for the pharmacokinetics and efficacy of protein biotherapeutics. Consequently, several analytical methods have been developed to evaluate the sialic acid content of glycoproteins. For example, antibody-based assays can be used to identify particular carbohydrate moieties.
Terminal sialic acid resides can be enzymatically detached from the glycoprotein of interest and analyzed by HPLC. However, each of these methods has shortcomings and typically requires pure samples or high concentrations. Conventional methods used by the biopharmaceutical industry suffer from poor accuracy, high data variability, and they cannot be used with complex culture media due to matrix interference. The method described herein overcomes such shortcomings and provides accurate and reproducible quantitation of protein sialylation.
Collectively, the method described herein comprises two steps: (1) an enzymatic reaction is used to hydrolyze the galactose and sialic acid residues from the glycoproteins; and (2) an ion-exchange chromatography method is used to separate and quantify the galactose residues. The enzymatic portion of the method involves the release of exposed (uncapped) terminal galactose residues by the specific exo-glycosidase, β-(1-4)-galactosidase (β-galactosidase), while the terminal sialic acid residues are released by α-(2-3,6,8,9)-sialidase (α-sialidase). Prior to digestion, a sample is divided among at least three tubes. The first tube, Reaction A, is a background sample, and comprises the enzyme reaction buffers only. The second tube, Reaction B, is reacted with β-galactosidase that cleaves all galactose residues that are not capped by sialic acids. The third tube, Reaction C, is co-digested with both neuraminidase and β-galactosidase. The neuraminidase enzyme removes the capping sialic acids and permits β-galactosidase to cleave all of the exposed galactose residues. High Performance Anion-exchange chromatography with Pulsed Amperometric Detection (HPAEC PAD) is then used to determine the amount of galactose present in the three samples. The ratio of uncapped galactose (i.e., Reaction B) to total galactose (i.e., Reaction C) is used to calculate percent capping of galactose residues, while also accounting for any free galactose present in the media (Reaction A).